Detection and analysis of circulating tumor cells (CTCs) have emerged as a promising way to diagnose cancer, study its cellular mechanism, and test or develop potential treatments. However, the rarity of CTCs among peripheral blood cells is a big challenge toward CTC detection. In addition, in cases where there is similar size range between certain types of CTCs (e.g. breast cancer cells) and white blood cells (WBCs), high‐resolution techniques are needed. In the present work, we propose a deterministic dielectrophoresis (DEP) method that combines the concept of deterministic lateral displacement (DLD) and insulator‐based dielectrophoresis (iDEP) techniques that rely on physical markers such as size and dielectric properties to differentiate different type of cells. The proposed deterministic DEP technology takes advantage of frequency‐controlled AC electric field for continuous separation of CTCs from peripheral blood cells. Utilizing numerical modeling, different aspects of coupled DLD‐DEP design such as the required applied voltages, velocities, and geometrical parameters of DLD arrays of microposts are investigated. Regarding the inevitable difference and uncertainty ranges for the reported crossover frequencies of cells, a comprehensive analysis is conducted on applied electric field frequency as design's determinant factor. Deterministic DEP design provides continuous sorting of CTCs from WBCs even with similar size and has the future potential for high throughput and efficiency.

We created an integrated microfluidic cell separation system that incorporates hydrophoresis and dielectrophoresis modules to facilitate high-throughput continuous cell separation. The hydrophoresis module consists of a serpentine channel with ridges and trenches to generate a diverging fluid flow that focuses cells into two streams along the channel edges. The dielectrophoresis module is composed of a chevron-shaped electrode array. Separation in the dielectrophoresis module is driven by inherent cell electrophysiological properties and does not require cell-type-specific labels. The chevron shape of the electrode array couples with fluid flow in the channel to enable continuous sorting of cells to increase throughput. We tested the new system with mouse neural stem cells since their electrophysiological properties reflect their differentiation capacity (e.g., whether they will differentiate into astrocytes or neurons). The goal of our experiments was to enrich astrocyte-biased cells. Sorting parameters were optimized for each batch of neural stem cells to ensure effective and consistent separations. The continuous sorting design of the device significantly improved sorting throughput and reproducibility. Sorting yielded two cell fractions, and we found that astrocyte-biased cells were enriched in one fraction and depleted from the other. This is an advantage of the new continuous sorting device over traditional dielectrophoresis-based sorting platforms that target a subset of cells for enrichment but do not provide a corresponding depleted population. The new microfluidic dielectrophoresis cell separation system improves label-free cell sorting by increasing throughput and delivering enriched and depleted cell subpopulations in a single sort.

While lipoplex (cationic lipid-nucleic acid complex)-mediated intracellular delivery is widely adopted in mammalian cell transfection, its transfection efficiency for suspension cells, e.g., lymphatic and hematopoietic cells, is reported at only ≈5% or even lower. Here, efficient and consistent lipoplex-mediated transfection is demonstrated for hard-to-transfect suspension cells via a single-cell, droplet-microfluidics approach. In these microdroplets, monodisperse lipoplexes for effective gene delivery are generated via chaotic mixing induced by the serpentine microchannel and co-confined with single cells. Moreover, the cell membrane permeability increases due to the shear stress exerted on the single cells when they pass through the droplet pinch-off junction. The transfection efficiency, examined by the delivery of the pcDNA3-EGFP plasmid, improves from ≈5% to ≈50% for all three tested suspension cell lines, i.e., K562, THP-1, Jurkat, and with significantly reduced cell-to-cell variation, compared to the bulk method. Efficient targeted knockout of the TP53BP1 gene for K562 cells via the CRISPR (clustered regularly interspaced short palindromic repeats)-CAS9 (CRISPR-associated nuclease 9) mechanism is also achieved using this platform. Lipoplex-mediated single-cell transfection via droplet microfluidics is expected to have broad applications in gene therapy and regenerative medicine by providing high transfection efficiency and low cell-to-cell variation for hard-to-transfect suspension cells.

Insulator-based dielectrophoresis (iDEP) is a powerful technique for separation and manipulation of bioparticles. In recent years, iDEP designs using arrays of insulating posts have shown promising results toward reaching high-efficiency bioparticle manipulation. Joule heating (JH) and electrothermal (ET) flows have been observed in iDEP microdevices and significantly affecting their performances. In this research, we utilize mathematical modeling to study, iDEP technique and the effects of JH and ET flow on device performance and propose a separation scenario for selective trapping of circulating tumor cells (CTCs). A robust numerical model is developed to calculate the distribution of electric and fluid flow fields in the presence of JH and ET flow, and predict the cells' trajectory inside the system. Our results indicate that JH not only induces temperature rise in the system, but also may alter the design iDEP separation scenario by inducing ET vortices that affect the cell's trajectory. To investigate the impact of JH-induced ET flow characteristics and vortex generation on separation efficiency, we introduce a dimensionless force ratio encompassing the effects of electrical field, drag forces, JH, and ET flow. Interestingly, it was found that ET flows can be used to significantly enhance the separation efficiency, even in higher inlet flow rates. Lastly, the effect of post geometry has been discussed.

Deterministic lateral displacement (DLD), which takes advantage of the asymmetric bifurcation of laminar flow around the embedded microposts, has shown promising capabilities in separating cells and particles of different sizes. Growing interest in utilizing highthroughput DLD devices for practical applications, such as circulating tumor cell separation, necessitates employing higher flow rates in these devices, leading to operating in moderate to high Reynolds number (Re) regimes. Despite extensive research on DLD devices in the creeping regime, limited research has focused on the physics of flow, critical size of the device, and deformable cell behavior in DLD devices at moderate to high Re. In this study, the transport behavior of particles/cells is investigated in realistic high-throughput DLD devices with hundreds of microposts by utilizing multiphysics modeling. A practical formula is proposed for the prediction of the device critical size, which could serve as a design guideline for high-throughput DLD devices. Then, the complex hydrodynamic interactions between a deformable cell and DLD post arrays are investigated. A dimensionless index is utilized for comparing different post designs to quantify the cell-post interaction. It is shown that the separation performances in high-throughput devices are highly affected by Re as well as the micropost shapes. These findings can be utilized for the design and optimization of high-throughput DLD microfluidic devices.